Butyric acid matrix metalloproteinase inhibitors

This application claims benefit of Provisional Application 60/024,025 filed Aug. 16, 1996.

This invention relates to a group of butyric acid derivatives which inhibit matrix metalloproteinase enzymes and thus are useful for treating diseases resulting from tissue breakdown, such as arthritis and osteoporosis.

Matrix metalloproteinases are naturally occurring enzymes found in most mammals and are associated with the breakdown of connective tissues. The class includes gelatinase A and B, stromelysin-1, fibroblast collagenase, neutrophil collagenase, matrilysin, and other forms of collagenase. These enzymes have been implicated with a number of diseases which result from breakdown of connective tissue, such as rheumatoid arthritis, osteoarthritis, osteoporosis, periodontitis, multiple sclerosis, gingivitis, corneal epidermal and gastric ulceration, atherosclerosis, neointimal proliferation which leads to restenosis and ischemic heart failure, and tumor metastasis. A method for preventing and treating these diseases is now recognized to be by inhibiting metalloproteinase enzymes, thereby curtailing and eliminating the breakdown of connective tissues that results in the disease states.

Several inhibitors of metalloproteinases have been identified. Many inhibitors are complex peptides, for instance as described by Chapman, et al., in J. Med. Chem., 1993;36:4293-4301. Small peptide inhibitors are also known, for example as described in U.S. Pat. Nos. 4,599,361 and 5,270,326, as well as nonpeptides as in WO 95/35276.

The need continues for small molecular weight molecules which can be economically prepared and yet are effective inhibitors of metalloproteinases. We have now discovered a group of butyric acid derivatives which have exceptionally good inhibitory activity. An object of this invention is to provide such compounds, their pharmaceutical formulations, and a method for using them to treat diseases mediated by metalloproteinases.

This invention provides butyric acid derivatives which are inhibitors of matrix metalloproteinases. The invention compounds have Formula I ##STR2## wherein:

X is O, NOR 9 , S, OH, SH, or ##STR3##

R 7 and R 7a independently are

hydrogen,

C 1 -C 20 alkyl or substituted C 1 -C 20 alkyl,

(CH 2 ) 0-6 -aryl,

(CH 2 ) 0-6 -heteroaryl, or

(CH 2 ) 0-6 -cycloalkyl;

R 1 and R 2 independently are

hydrogen,

C 1 -C 20 alkyl or substituted C 1 -C 20 alkyl,

halo,

NO 2 ,

CN,

CHO,

COR 6 ,

COOR 6 ,

SO 3 R 6 ,

OR 6 ,

CONR 4 R 5 ,

(CH 2 ) 0-6 -aryl,

(CH 2 ) 0-6 -heteroaryl, or

(CH 2 ) 0-6 -cycloalkyl;

R 6 is hydrogen,

C 1 -C 20 alkyl or substituted C 1 -C 20 alkyl;

aryl is phenyl or substituted phenyl;

R 3 is hydroxy,

O--C 1 -C 20 alkyl or substituted O--C 1 -C 20 alkyl,

O--(CH 2 ) 1-3 aryl, or

NHOR 6 ;

R 4 and R 5 independently are hydrogen,

C 1 -C 20 alkyl or substituted C 1 -C 20 alkyl,

C 2 -C 20 alkenyl or substituted C 2 -C 20 alkenyl,

(CH 2 ) 0-6 -aryl,

(CH 2 ) 0-6 -(O or S)-aryl,

(CH 2 ) 0-6 -heteroaryl,

(CH 2 ) 0-6 -(O or S)-heteroaryl;

or one of R 4 and R 5 is hydrogen and the other is:

COR 8 ,

CSR 8 ,

CONR 8 R 9 ,

CSNR 8 R 9 ,

COOR 8 ,

COSR 8 , ##STR4## S(O) 1 or 2 -C 1 -C 20 alkyl or substituted alkyl, S(O) 1 or 2 -aryl

S(O) 1 or 2 -heteroaryl, or

S(O) 1 or 2 -cycloalkyl;

Y is ##STR5##

R 8 and R 9 independently are

hydrogen,

C 1 -C 20 alkyl or substituted C 1 -C 20 alkyl,

C 2 -C 20 alkenyl or substituted C 2 -C 20 alkenyl,

(CH 2 ) 0-6 -(O or S) 0-1 -aryl,

(CH 2 ) 0-6 -(O or S) 0-1 -heteroaryl, or

(CH 2 ) 0-6 -(O or S) 0-1 -cycloalkyl;

W, W 1 , Z, and Z 1 independently are CR 1 or N;

the term ##STR6## is a double bond when X is O, S, NOR 9 , or

N-NR 7 R 7a , and a single bond when X is OH or SH; and the pharmaceutically acceptable salts, isomers, stereoisomers, and solvates thereof.

Preferred compounds are defined by Formula II ##STR7## wherein R 1 , R 2 , R 3 R 4 , R 5 , and X are as defined above.

Further preferred compounds have the formula ##STR8## and especially preferred are those wherein X is O, and R 3 is hydroxy. The most preferred compounds of the invention are in this latter group where R 4 is hydrogen, and R 5 is COR 8 , COOR 8 , or CONR 8 R 9 , where R 8 and R 9 are alkyl or substituted alkyl, aryl or substituted aryl.

A further embodiment of this invention is a pharmaceutical formulation comprising a compound of Formula I admixed with a pharmaceutically acceptable carrier, excipient, or diluent therefor.

The invention additionally provides a method for inhibiting the hydrolytic activity of a matrix metalloproteinase enzyme comprising administering to a mammal an effective amount of a compound of Formula I. In a preferred embodiment, the invention is a method for inhibiting or treating multiple sclerosis, atherosclerotic plaque rupture, aortic aneurysms, heart failure, restenosis, periodontal disease, corneal ulceration, cancer metastasis, tumor angiogenesis, arthritis, and for wound healing.

As used herein, the terms "C 1 -C 20 alkyl" and "substituted C 1 -C 20 alkyl" mean straight or branched aliphatic groups having from 1 to 20 carbon atoms, preferably 1 to 12, and typically 1 to 6, optionally substituted. Exemplary alkyl groups include methyl, ethyl, isopropyl, 1,1-dimethylheptyl, n-decyl, 1-n-butyl-2-isopropyldecyl, tert-butyl, isoundecyl, and the like. Substituted alkyl groups are the foregoing C 1 -C 20 alkyl groups having one or more, typically from 1 to 3, substituent groups such as halo, hydroxy, C 1 -C 4 alkoxy, thio, C 1 -C 4 alkylthio, phenyl, alkylphenyl, phenoxy, alkylphenoxy, substituted phenyl or substituted phenoxy. Typical "substituted C 1 -C 20 alkyl" groups thus include hydroxymethyl, n-butoxymethyl, chloromethyl, 1-(3-chlorophenoxy)ethyl-, 1,2,4-trifluorohexyl, 1-(phenoxymethyl)ethyl, 2-methoxydecyl, 4-methylthio-5-bromo-undecyl, 1-ethyl-3-phenylbutyl, 4-(3,4-dibromophenyl)-heptyl, pentadecafluoro-octyl, and 2,2,2-trifluoroethyl.

The terms "C 2 -C 20 alkenyl" and "substituted C 2 -C 20 alkenyl" mean the foregoing alkyl groups having one to four non-adjacent double bonds. Examples include prop-2-enyl, but-2-enyl, oct-2,4-dienyl, 2-phenylethenyl, 3-naphthyl-but-2-enyl, and 6-(2-pyridyl)-hex-3-enyl.

The term "halo" includes fluoro, bromo, chloro, and iodo. Several of the R groups in Formula I can be a group defined as "COR 6 ", "COOR 6 ", "SO 3 R 6 ", and "OR 6 ", where R 6 is hydrogen, alkyl, or substituted alkyl as those terms are defined above. The COR 6 groups thus include formyl, acetyl, propionyl, pivaloyl, 3-hydroxybutyryl, undecanoyl, 4-n-butyl-5-chloro-6-methylthio-heptanoyl, 4-bromophenoxyacetyl, and 4-phenylpentanoyl. Typical COOR 6 groups include methoxycarbonyl, benzyloxycarbonyl, n-octyloxycarbonyl, 5-methoxy-6-thioheptyloxycarbonyl, and the like. Typical SO 3 R 6 groups include methoxysulfonyl, 3-chlorobenzyloxysulfonyl, and 4-chlorobutyloxy-sulfonyl. The OR 6 groups include methoxy, tert-butoxy, benzyloxy, 4-nitrobenzyloxy, and the like.

The term "CONR 4 R 5 " means an aminocarbonyl substituent such as methylaminocarbonyl, benzylaminocarbonyl, diethylaminocarbonyl, 3-pyridylmethylaminocarbonyl, and the like.

The term "aryl" means a monocyclic or bicyclic aromatic hydrocarbyl group, for example, phenyl or naphthyl, optionally substituted with from one to five groups, typically one, two, or three, selected from halo, hydroxy, methylenedioxy, phenyl, nitro, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, thio, C 1 -C 6 alkylthio, amino, C 1 -C 6 alkyl and dialkylamino, CHO, COR 6 , COOR 6 , OR 6 , SO 3 R 6 , or CONR 4 R 5 . Typical (CH 2 ) 0-6 -aryl groups thus include phenyl, 3-hydroxyphenyl, 2-methylphenyl, 4-chlorobenzyl, 3,4-methylenedioxyphenyl, 4-(2-methylthiophenyl)butyl, 2-(3-aminophenyl)ethyl, 6-(2-chloro-3-methylamino-4-formylphenyl)hexyl, naphthyl, 3-chloronaphthyl, and 2,4,6-trimethyl-naphthyl.

The term "(CH 2 ) 0-6 -(O or S)-aryl" means the foregoing aryl groups bonded through oxygen or sulfur. Examples include phenoxymethyl, 2-phenylthioethyl, l-naphthyloxymethyl, and 3-bromo-l-naphthylthiomethyl.

The term "(CH 2 ) 0-6 -heteroaryl" means a heteroaryl group bonded directly or through from one to six methylene groups. The term "heteroaryl" means a monocyclic or fused bicyclic aromatic ring having from 4 to 12 carbon atoms and from 1 to 3 heteroatoms selected from O, S, or N. The heteroaryl group can be substituted with the same groups as indicated above for phenyl. Typical heteroaryl groups thus include pyridyl, 2- or 3-furanyl, 2- or 3-thienyl, 2- or 3-pyranyl, 2-, 4-, or 5-pyrimidinyl, 3- or 4-pyridazinyl, 2- or 3-morpholinyl, indolyl, 3-phenyl-2-pyridyl, benzoxazolyl, benzopyranyl, quinolinyl, quinoxalinyl, isoquinolinyl, and pyridopyrimidinyl. Substituted heteroaryl groups include 3-chlorothiophene, 3-hydroxyfuryl, and 3-nitromorpholine.

The term "(CH 2 ) 0-6 -(O or S)-heteroaryl" means the foregoing heteroaryl groups bonded through oxygen or sulfur- Examples include 3-pyridyloxymethyl, 2-(2-pyranyl)oxyethyl, and 3-furanylthiomethyl.

The term "(CH 2 ) 0-6 -(O or S) 0-1 -cycloalkyl" means a cycloalkyl group bonded directly or through from one to six methylene groups, and optionally through oxygen or sulfur. "Cycloalkyl" means a monocyclic or bicyclic carbocyclic group having from three to ten carbon atoms, and optionally substituted with one, two, or three groups selected from halo, hydroxy, phenyl, nitro, cyano, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, thio, C 1 -C 6 alkylthio, amino, C 1 -C 6 alkyl and dialkylamino, CHO, COR 6 , COOR 6 , OR 6 , SO 3 R 6 , and CONR 4 R 5 . Typical cycloalkyl groups thus include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-ethylcyclopentyl, 3,4-dichlorocyclohexyl, decalinyl, adamantyl, 3-aminocyclooctyl, and 2-phenylcyclohexyl.

R 4 and R 5 are nitrogen substituents and include hydrogen, alkyl and substituted alkyl, (CH 2 ) 0-6 -(O or S) 0-1 -aryl and (CH 2 ) 0-6 -(O or S) 0-1 -heteroaryl, as those terms are defined above. Preferred compounds of the invention are those wherein R 4 is hydrogen, and R 5 is other than hydrogen, especially COR 8 , CSR 8 , CONR 8 R 9 , CSNR 8 R 9 , COOR 8 , SO 2 -alkyl, SO 2 -aryl, and COSR 8 . These groups define compounds which are, respectively, amides, thioamides, ureas, thioureas, carbamates, sulfonamides, and thiocarbamates. Typical "COR 8 " groups thus include formyl, acetyl, bromoacetyl, trifluoroacetyl, cyclobutylformyl, 3-nitropropionyl, 4-methoxycarbonylbutyryl, benzoyl, 4-phenylbenzoyl, phenoxyacetyl, l-phenoxyethyl, 6-dimethylaminocarbonyl-octanoyl, and 4-phenyldecanoyl. Typical "CSR 8 " groups include methylthiocarbonyl, ethylthiocarbonyl, and tert-butylthiocarbonyl. Examples of "CONR 8 R 9 " groups include aminocarbonyl, dimethylaminocarbonyl, dibenzylaminocarbonyl, and the like. The invention includes thioureas, i.e., compounds of Formula I where R 4 is hydrogen, and R 5 is ##STR9## Typical of such R 5 groups are aminothiocarbonyl, diethylaminothiocarbonyl, and benzylaminothiocarbonyl. The invention also includes sulfonamides, for example when R 5 is --SO 2 alkyl or --SO2 aryl. Typical groups include isopropylsulfonyl, phenylsulfonyl, naphthylsulfonyl, and 4-chlorophenylsulfonyl.

R 4 and R 5 can include "COOR 8 " and "COSR 8 "; preferably, one of R 4 or R 5 is hydrogen. Such groups form carbamates and thiocarbamates, respectively. Typical COOR 8 groups include methoxycarbonyl, 3-hydroxybutoxycarbonyl, benzyloxycarbonyl, 2,4-dibromobenzyloxycarbonyl, l-methoxy-2-phenyl-3-chloropropoxycarbonyl, and 10-methoxyundecyloxycarbonyl. Examples of "COSR 8 " groups include methylthiocarbonyl, benzylthiocarbonyl, and the like.

Ideally, one of R 4 and R 5 is hydrogen when the other is ##STR10## These groups are residues of naturally occurring as well as unnatural amino acids, for example, glycyl, alanyl, N-methylvalyl, N-benzylleucyl, N,N-dimethylphenylalanyl, threonyl, arginyl, glutamyl, lysyl, tyrosyl, 2-amino-6-chlorohexanoyl, 2-benzylamino-3,4-dimethoxyheptanoyl, and the like.

Ideally, R 4 is hydrogen when R 5 is a group having the formula ##STR11## Examples of these groups include methylaminoformamidocarbonyl, ethoxyformamidocarbonyl, methylthioformamidocarbonyl, diethylaminosulfonylaminocarbonyl, and N-benzyloxysulfonyl-N-ethylaminocarbonyl.

Typical compounds provided by this invention are illustrated by the following general formulas: ##STR12##

Especially preferred compounds are those illustrated below: ##STR13## where R 10 is:

--CF 3 , ##STR14##

C 1 -C 12 alkyl, or perfluoro-C 1 -C 12 alkyl, ##STR15##

The invention compounds of Formula I are prepared by any of several procedures, utilizing reaction processes well-known to those having ordinary skill in the art of organic chemistry. A typical invention compound can be prepared by acylating a tricyclic reactant with an amino substituted butyryl acylating agent such as aspartic acid anhydride, or a butyryl agent of the formula ##STR16## where L is a leaving group such as chloro or formyloxy, and R 3 , R 4 , and R 5 are as defined above. The acylation is conducted under Friedal Crafts conditions, for example utilizing aluminum chloride as a catalyst, and conducting the reaction in an organic solvent, preferably a halogenated hydrocarbon such as dichloromethane or chloroform. A preferred process comprises reacting a tricyclic starting material with an N-substituted D- or L-aspartic acid anhydride according to the following general scheme: ##STR17## The reactants are combined in approximately equimolar quantities in a solvent such as dichloromethane with an equivalent or excess of aluminum chloride. The acylation typically is complete within about 8 to about 24 hours when carried out at a temperature of about -5° C. to about 20° C. The product is readily isolated by pouring the reaction mixture into ice/water, and separating the organic layer. Removal of the solvent by evaporation affords the invention compound, which can be purified by standard techniques such as chromatography and crystallization.

The compound thus prepared can be converted to other invention compounds (i.e., where one or both of R 4 and R 5 are other than hydrogen, or where the 4-oxo group is converted to an oxime) by conventional means. For example, a compound of the above formula wherein R 4 is hydrogen, and R 5 is an acyl group such as trifluoroacetyl readily reacts with ammonia in an alcohol such as methanol to afford the corresponding primary amine, where both R 4 and R 5 are hydrogen. The primary amine can be acylated or alkylated by conventional means, for example by reaction with an acyl halide such as acetyl chloride or glycyl bromide, a sulfonyl halide such as isopropylsulfonyl chloride or benzylsulfonyl bromide, an anhydride such as acetic anhydride or acetic-formic anhydride, or an alkyl halide such as methyl bromide or ethyl iodide, to provide the corresponding N-acyl or N-alkyl derivative. The butanoic acid esters of the invention, i.e., where R 3 is alkoxy or substituted alkoxy, are readily prepared by reacting the free acid of the above formula with an alcohol under acidic conditions, or by other conventional esterification techniques. Such reactions are depicted in Scheme 1. ##STR18##

The butyric acid esters of Formula I, compounds wherein R 3 is O-alkyl or O-substituted alkyl, are especially useful as intermediates, in that other sites in the molecule can be derivatized, and the ester function can subsequently be hydrolyzed to provide the free acid. The ester groups are readily hydrolyzed to the corresponding carboxylic acids by routine methods, for instance by reaction with a strong acid such as trifluoroacetic acid, polyphosphoric acid, sulfuric acid, or the like. The hydrolysis generally is carried out at a temperature of about 0° C. to about 25° C., and normally is complete within about 2 to 24 hours. The product, a compound of Formula I wherein R 3 is OH, can be isolated by diluting the reaction mixture with water and extracting the product into a water immiscible solvent such as ethyl acetate, dichloromethane, or the like, and then removing the organic solvent, for example by evaporation under reduced pressure. The free carboxylic acids thus formed can be converted to salts by reaction with a base such as sodium hydroxide, calcium carbonate, or the like. The carboxylic acids also can be reacted with hydroxylamine hydrochloride to form the corresponding hydroxamic acids, i.e., compounds of Formula I where R 3 is NHOH.

The invention compounds contain at least one asymmetric carbon atom, and as such exist as optically active isomers. The invention contemplates the racemic forms as well as the individual isomers. The individual isomers can be prepared from optically pure starting materials, for example by utilizing naturally occurring amino acids, or by resolving the racemate by normal techniques such as chromatography and the like.

Synthesis of various invention compounds of Formula I can be facilitated by utilizing common protecting groups on functional groups such as hydroxy, carboxy, and amino. Protecting groups will be utilized to prevent unwanted side reactions and are readily removed when desired to provide the invention compound. The use of protecting groups is well-documented, for example, by Greene and Wuts in "Protective Groups in Organic Synthesis," 2nd Ed., 1991, John Wiley & Sons, Inc. Typical carboxy acid and hydroxy protecting groups are readily removable ester and ether forming groups such as 2,2,2-trichloroethyl, benzyl, methyl, trimethylsilyl, acetyl, and the like. Primary amino groups, for example, compounds of Formula I wherein R 4 and R 5 both are hydrogen, are readily protected with common acyl groups such as acetyl, pivaloyl, trifluoroacetyl, trimethylsilyl, and t-butyldimethylsilyl.

A key intermediate for the synthesis of most of the compounds of this invention is the primary amines of Formula I wherein R 4 and R 5 both are hydrogen. These compounds are prepared as described above and in Scheme 1. In a preferred embodiment, the key intermediate has the formula ##STR19## where R 1 , R 2 , and R 3 are as defined above. Such primary amino compounds readily undergo standard reactions of amines to provide invention compounds of Formula I wherein one or both of R 4 and R 5 are other than hydrogen. For example, the primary amines react with acylating agents such as acid halides and acid anhydrides to produce the corresponding amides. Typical acid halides that can be utilized include ##STR20## and typical anhydrides include ##STR21## Ureas, e.g., Formula I where R 4 is hydrogen and R 5 is CONR 8 R 9 , can be prepared by reacting the primary amine with an isocyanate of the formula R 8 --N═C═O, followed by alkylation or acylation to afford compounds wherein R 9 is other than hydrogen. Carbamates and thiocarbamates, e.g., Formula I where R 4 is hydrogen and R 5 is ##STR22## respectively, are prepared by reacting the primary amine with acid halides of the formula ##STR23## Similar acylations can be carried out with acid halides such as ##STR24## The primary amines (Formula I where R 4 and R 5 both are H) can also be reacted with chlorocarbonyl isocyanate to give an acid chloride intermediate that can then be reacted with amines, alcohols, and thiols to provide invention compounds. The reaction of the amine and chlorocarbonyl isocyanate is depicted as follows: ##STR25## The primary amine and chlorocarbonyl isocyanate generally are mixed in equimolar quantities in an unreactive organic solvent such as diethyl ether or dichloromethane. The reaction generally is complete within about 1 to 2 hours when carried out at about 0° C. The intermediate acid chloride readily reacts with amines, alcohols, and thiols according to the following scheme: ##STR26## The primary amine (Formula I where R 4 and R 5 both are hydrogen) can also be reacted with chlorosulfonyl isocyanate to provide a sulfonyl chloride: ##STR27## The sulfonyl chloride can be reacted with an amine or alcohol as follows: ##STR28## The primary amines can additionally be acylated by reaction with a carboxy acid in the presence of a peptide coupling agent. Typical coupling agents include N,N'-dicyclohexycarbodiimide, N,N'-diisopropylcarbodiimide, N,N'-diethylcarbodiimide, and 1,2-dihydroquinoline.

As noted above, the invention includes pharmaceutically acceptable salts and solvates of compounds having Formula I. The carboxylic acids (i.e., R 3 is OH) readily react with inorganic and organic bases to form such salts. Typical inorganic bases commonly employed include sodium hydroxide, potassium carbonate, calcium phosphate, and sodium bicarbonate. Organic bases routinely used include diethylamine, pyridine, benzylamine, triethanolamine, morpholine, and ethylenediamine. Compounds of Formula I which have a basic group, for example when R 4 and R 5 both are hydrogen, can form acid addition salts by reaction with inorganic or organic acids. Typical acids include hydrochloric acid, sulfuric acid, phosphoric acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid and the like. The salts of the invention typically are highly crystalline under normal conditions and can be purified by crystallization from common solvents such as ethyl acetate, chloroform, ethanol, water, toluene, diethyl ether, and hexane. Such crystallizations can produce the invention compound as a solvate, for example as a hydrate or ethanolate, which forms can be utilized in pharmaceutical preparations.

The synthesis of invention compounds is further illustrated in Scheme 2, as well as in the following detailed examples. The examples are illustrative only and are not intended to be limiting in any respect. ##STR29##

No. Compound (m + 1)

______________________________________

18 4-Dibenzofuran-2-yl-2-

478

diphenylacetylamino-4-oxo-butyric

acid

19 2-[2-(4-Chloro-phenoxy)- 452

acetylamino]-4-dibenzofuran-2-yl-4-

oxo-butyric acid

20 4-Dibenzofuran-2-yl-2-(3,4- 456

dichloro-benzoylamino-4-oxo-butyric

acid

21 4-Dibenzofuran-2-yl-2-[2-(3,4- 462

dimethoxy-phenyl)-acetylamino] -4-

oxo-butyric acid

22 4-Dibenzofuran-2-yl-2- --

[(naphthalene-2-carbonyl)-amino] -4-

oxo-butyric acid

23 4-Dibenzofuran-2-yl-2-(2,2- 396

dimethyl-pentanoylamino)-4-oxo-

butyric acid

24 4-Dibenzofuran-2-yl-4-oxo-2- --

[(pyridine-4-carbonyl)-amino]-

butyric acid

25 4-Dibenzofuran-2-yl-4-oxo-2-(3- 414

phenyl-acryloylamino)-butyric acid

26 2-[(Adamantane-1-carbonyl)-amino]- --

4-dibenzofuran-2-yl-4-oxo-butyric

acid

27 4-Dibenzofuran-2-yl-4-oxo-2- --

(2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-

pentadecafluoro-octanoylamino)-

butyric acid

28 4-Dibenzofuran-2-yl-4-oxo-2-(2- 418

phenoxy-acetylamino)-butyric acid

29 4-Dibenzofuran-2-yl-2-(4-methyl- 402

benzoylamino)-4-oxo-butyric acid

30 4-Dibenzofuran-2-yl-4-oxo-2-(2- 432

phenoxy-propionylamino)-butyric

acid

31 2-[4-(4-Bromo-phenyl)- 508

butyrylamino]-4-dibenzofuran-2-yl-

4-oxo-butyric acid

32 2-(2-Benzyloxy-acetylamino)-4- 432

dibenzofuran-2-yl-4-oxo-butyric

acid

33 4-Dibenzofuran-2-yl-2- 396

heptanoylamino-4-oxo-butyric acid

34 2-(4-Butyl-benzoylamino)-4- 444

dibenzofuran-2-yl-4-oxo-butyric

acid

35 2-(4-Cyano-benzoylamino)-4- 413

dibenzofuran-2-yl-4-oxo-butyric

acid

36 2-[(3-Chloro-thiophene-2-carbonyl)- 428

amino)-4-dibenzofuran-2-yl-4-oxo-

butyric acid

37 2-(4-Butoxy-benzoylamino)-4- 460

dibenzofuran-2-yl-4-oxo-butyric

acid

38 2-(Cyclopropanecarbonyl-amino)-4- 352

dibenzofuran-2-yl-4-oxo-butyric

acid

39 2-(4-Chloro-benzoylamino)-4- 422

dibenzofuran-2 -yl-4-oxo-butyric

acid

40 4-Dibenzofuran-2-yl-4-oxo-2- --

(2,3,4,5,6-pentafluoro-

benzoylamino)-butyric acid

41 4-Dibenzofuran-2-yl-2-[(furan-2- 378

carbonyl)-amino]-4-oxo-butyric acid

42 2-(4-Bromo-benzoylamino)-4- 466

dibenzofuran-2-yl-4-oxo-butyric

acid

43 4-Dibenzofuran-2-yl-2-(4-fluoro- 406

benzoylamino)-4-oxo-butyric acid

44 4-Dibenzofuran-2-yl-4-oxo-2- 440

[(quinoxaline-2-carbonyl)-amino]-

butyric acid

45 4-Dibenzofuran-2-yl-4-oxo-2- 389

[(pyridine-2-carbonyl)-amino]-

butyric acid

46 4-Dibenzofuran-2-yl-2-(4-methoxy- 418

benzoylamino)-4-oxo-butyric acid

47 4-Dibenzofuran-2-yl-2-(3,4- 448

dimethoxy-benzoylamino)-4-oxo-

butyric acid

48 4-Dibenzofuran-2-yl-4-oxo-2-(4- 456

trifluoromethyl-benzoylamino)-

butyric acid

49 2-(Cyclopentanecarbonyl-amino)-4- 380

dibenzofuran-2-yl-4-oxo-butyric

acid

50 2-(Cyclohexanecarbonyl-amino)-4- 394

dibenzofuran-2-yl-4-oxo-butyric

acid

51 4-Dibenzofuran-2-yl-4-oxo-2- 394

[(thiophene-2-carbonyl)-amino]-

butyric acid

52 4-Dibenzofuran-2-yl-4-oxo-2-(2- --

thiophen-2-yl-acetylamino)-butyric

acid

53 2-[(Benzo[1,3)dioxole-5-carbonyl)- 432

amino]-4-dibenzofuran-2-yl-4-oxo-

butyric acid

54 4-Dibenzofuran-2-yl-4-oxo-2-(2- 430

phenylbutyrylamino)-butyric acid

55 4-Dibenzofuran-2-yl-2-[(5-methyl-2- 469

phenyl-2H-[1,2,3]triazole-4-

carbonyl)-amino]-4-oxo-butyric acid

______________________________________

The invention compounds have been evaluated in standard in vitro assays and shown to be potent inhibitors of several matrix metalloproteinase enzymes. The assays measure the amount by which a test compound reduces the hydrolysis of a thiopeptolide substrate caused by a matrix metalloproteinase enzyme. Such assays are described in detail by Ye, et al., in Biochemistry, 1992;31(45):11231-11235).

Thiopeptolide substrates show virtually no decomposition or hydrolysis in the absence of a matrix metalloproteinase enzyme. A typical thiopeptolide substrate commonly utilized for assays is Ac-Pro-Leu-Gly-thioester-Len-Len-Gly-O Et. A 100 μL assay mixture will contain 50 mM of 2-morpholinoethane sulfonic acid monohydrate (MES, pH 6.0) 10 mM CaCl 2 , 100 μM thiopeptolide substrate, and 1 mM 5,5'-dithio-bis-(2-nitro-benzoic acid (DTNB). The thiopeptolide substrate concentration is varied from 10 to 800 μM to obtain Km and Kcat values. The change in absorbance at 405 nm is monitored on a Thermo Max microplate reader (molecular Devices, Menlo Park, Calif.) at room temperature (22° C.). The calculation of the amount of hydrolysis of the thiopeptolide substrate is based on E 412 =13600 m -1 cm -1 for the DTNB-derived product 3-carboxy-4-nitrothiophenoxide. Assays are carried out with and without matrix metalloproteinase inhibitor compounds, and the amount of hydrolysis is compared for a determination of inhibitory activity of the test compounds.

Several representative compounds have been evaluated for their ability to inhibit various matrix metalloproteinase enzymes. Table I below presents inhibitory activity for several invention compounds. In the table, MMP-1 refers to interstitial collagenase; MMP-2 refers to Gelatinase A; MMP-3 refers to stromelysin; MMP-7 refers to matrilysin; and MMP-13 refers to Collagenase 3. Test compounds were evaluated at various concentrations in order to determine their respective IC 50 values, the micromolar concentration of compound required to cause a 50% inhibition of the hydrolytic activity of the respective enzyme.

______________________________________

(IC.sub.50) μM

Compound of

______________________________________

1 100 0.059 0.95 100 2.8

2 -- 44.0 78 -- --

3 -- 3.8 33 -- --

4 -- 0.16 1.6 -- --

5 -- 0.32 1.7 -- --

6 -- 0.18 0.91 -- --

7 -- 0.084 0.33 -- --

8 100 0.067 0.29 100 1.3

9 -- 0.51 3.9 -- --

10 -- 1.64 9.4 -- --

11 100 0.36 2.7 100 6.8

12 100 8.8 4.0 -- --

13 -- 0.28 9.2 100 --

14 83 0.017 0.26 100 1.1

15 100 12.5 76.5 100 100

16 100 0.72 6.6 100 2.4

18 100 0.1 0.36 100 1.1

19 100 0.64 3.1 100 46

20 100 1.3 7.0 100 38

21 100 0.32 2.4 100 16

22 100 1.7 6.9 100 28

23 100 0.17 0.76 100 5.6

24 100 3.2 13.0 100 41

25 100 0.11 0.8 100 8.4

26 100 2.1 9.2 100 40

27 100 4.6 18 100 77

28 100 0.18 0.51 100 12

29 100 0.05 0.39 100 5.2

30 45 0.035 0.1 100 2.9

31 100 0.6 5.3 100 25

32 100 0.041 0.33 100 3.5

33 100 0.06 0.64 100 4.7

34 100 0.87 5.4 100 49

35 100 0.11 1.1 100 11

36 100 0.14 1.0 100 13

37 100 0.8 5.0 100 58

38 100 0.09 0.85 100 5.6

39 100 0.15 1.2 100 12

40 100 5.2 28 100 100

41 100 0.14 1.5 100 12

42 100 0.18 1.3 100 17

43 100 0.07 0.66 100 7.1

44 100 0.31 3.5 100 27

45 100 0.11 1.0 100 15

46 100 0.14 1.0 100 11

47 100 0.13 0.87 100 8.8

48 100 0.32 1.5 100 20

49 100 0.08 0.35 100 4.4

50 100 0.15 0.7 100 9.1

51 100 0.16 1.3 100 16

52 100 2.5 7.7 100 48

53 100 0.13 1.0 100 9.2

54 100 0.06 0.25 100 1.5

55 100 1.8 14 100 70

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The compounds were additionally evaluated for their ability to inhibit full-length collagenase hydrolysis of thiopeptolide substrate (FLC) and full-length gelatinase B (FLGB) hydrolysis of thiopeptolide. The results of representative compounds is given in Table II.

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(IC.sub.50) μM

Compound of

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1 >100 >100

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The compounds of the present invention can be prepared and administered in a wide variety of oral and parenteral dosage forms. Thus, the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. Also, the compounds of the present invention can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. It will be obvious to those skilled in the art that the following dosage forms may comprise as the active component, either a compound of Formula I or a corresponding pharmaceutically acceptable salt of a compound of Formula I. The active compound generally is present in a concentration of about 5% to about 95% by weight of the formulation.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component, with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing, and thickening agents as desired.

Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

The pharmaceutical preparation is preferably in unit dosage form. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The quantity of active component in a unit dose preparation may be varied or adjusted from 1 mg to 1000 mg, preferably 10 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.

In therapeutic use as agents for the treatment of multiple sclerosis, atherosclerotic plaque rupture, aortic aneurism, heart failure, restenosis, periodontal disease, corneal ulceration, cancer metastasis, tumor angiogenesis, arthritis, or other autoimmune or inflammatory disorders dependent upon breakdown of connective tissue, the compounds utilized in the pharmaceutical method of this invention are administered at a dose that is effective to inhibit the hydrolytic activity of one or more matrix metalloproteinase enzymes. The initial dosage of about 1 mg to about 100 mg per kilogram daily will be effective. A daily dose range of about 25 mg to about 75 mg per kilogram is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstance is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. Typical dosages will be from about 0.1 to about 500 mg/kg, and ideally about 25 to about 250 mg/kg.

The following examples illustrate typical formulations provided by the invention.